Annually in the U.S., aneurysmal subarachnoid hemorrhage affects greater than 30,000 people. Ten to 15 percent of these individuals die before reaching the hospital and greater than 50 percent die within the first month following rupture. Of those patients that survive, approximately half suffer some permanent neurological deficit.
Intra-cranial saccular (berry) aneurysm is a balloon-like distension of a major brain artery occurring at (or near) the apex of arterial forks. It is frequently (90%) located on the anterior part of the circle of Willis. Various hypothesis have been proposed regarding the developmental mechanisms of Saccular Cerebral Artery Aneurysms (SCAAs) such as the medial defect theory (Forbus, 1930), the elastic lamellar theory (Glynn, 1940), degenerative theory (Stehbens, 1963; Stehbens, 1972), congenital theories (Bremer, 1943; Agnoli, 1982), and others (Sekha et al., 1981). Recently the development of an experimental animal model of the disease with pathological features very similar to those of human SCCA has made possible the study of the pathogenesis of human SCCAs (Hashimoto et al., 1978). It has been shown that hemodynamic stress induces the development of cerebral aneurysms causing degenerative changes of the endothelium, the elastic lamina and the medial smooth muscle cells at specific site on the arterial bifurcation (Kojima et al., 1988).
Histological Features of SCAAs
The anterior cerebral artery/olfactory artery (ACA/OA) junction is one of the most common sites of aneurysm development in the animal model. Its normal structure and changes due to aneurysm development have been widely studied.
1. Normal ACA/OA Artery Junction
The apex of a normal ACA/OA junction consists of normal arterial components (endothelial cells, internal elastic lamina, medial smooth muscle cells, and thin adventitial fibrous connective tissue). In the apical region, there is an intimal protrusion called pad consistently located near the apex on the distal side of the ACA. This pad is composed of spindle-shaped cells similar to the medial smooth muscle cells, rich in interstitial tissue. Under and just distal to the intimal pad on the side of the ACA, the internal elastic lamina is thinned and fragmented.
2. Aneurysm Formation at ACA/OA Artery Junction
The initial changes of aneurysm occur at the intimal pad and the neighboring distal portion. In the histological structure of early stage aneurysms, there are the following characteristics: 1) the wall does not significantly protrude; 2) initial changes are localized almost exclusively at the intimal pad and its neighboring distal portion; and 3) there is fragmentation of internal elastic lamina and slight thinning of the media (decrease of medial smooth muscle cells in number). In the histological structure of advanced stage aneurysms, there are the following characteristics: 1) aneurismal wall consists only of a fibrous adventitia and a layer of endothelial cells; 2) complete disappearance of the Internal Elastic Lamina (I.E.L) at the level of the aneurismal neck; and 3) media layer (smooth muscle cells (SMC)) ceases abruptly proximal to the neck.
The histological features of SCAAs include degenerative changes of endothelium, fragmentation and disappearance of I.E.L, and thinning (then disappearance) of medial layer. In degenerative changes of the endothelium, the following has been observed. Severe changes in endothelium have been reported. Nagata et al. (1981) examined by scanning electron microscopy the luminal surface of the cerebral aneurysms. They noticed some variations in the shape of the endothelial cells from fusiform to polygonal. Some of them showed balloon-like protrusions. Crater-like depressions on the endothelial surface and small holes and enlarged gaps at the junction of the endothelial cells were frequently observed. Gap formation at the junctions between the endothelial cells was one of the most obvious changes on the luminal surface of the aneurysms. Kojima et al. (1986) studying various stage of early aneurismal changes reported alterations of the endothelium developing just distal to intimal pad. Degenerated cells with balloons and craters were observed intermingled with regenerated endothelial cells. Interendothelial gaps were also seen. They concluded that some hemodynamic stress, possibly turbulent flow or secondary flow, may injure the endothelial cells located distal to the pad, and such injured endothelial cells in turn develop saccular cerebral aneurysms. Greenhill and Stehbens (1982) also described severe alterations of the endothelium and subendothelial tissues caused by hemodynamic stress. Kim et al. (1992) studied aneurismal changes in experimental monkeys and found endothelial injury. They suggested that aneurismal changes are initiated by degenerative changes in the endothelium, which are followed by alterations in the underlying elastic lamina and, in turn, in the medial layer.
Degenerative changes of the internal elastic lamina and the medial smooth muscle cells are also known. Hazama et al. (1986) showed that early aneurismal changes consist on degenerative changes of the Internal Elastic Lamina (I.E.L) at the intimal pad and the neighboring area distal to the pad associated to regressive changes of medial smooth muscle layer. Kim et al. (1988) also reported degenerative changes of the I.E.L and medial smooth muscle layer. Morimoto et al. (2002) found that the characteristic of SCAA formation in a mouse model was thinning of medial smooth muscle layer and disappearance of the I.E.L. Kondo et al. (1998) found that the histological features of aneurismal changes were thinning of the medial layer accompanied by fragmentation or disappearance of internal elastic lamina with wall dilatation. They noted a decreased number of SMCs in the medial layer due to apoptosis. They concluded that the death of medial SMCs through apoptosis plays an important role in aneurysm formation.
Molecular Mechanisms of SCAAs Formation
While the pathological features of aneurismal lesions described above are well documented, the precise molecular mechanisms involved in the formation of cerebral aneurysms have not yet been conclusively identified. Hemodynamic stress has been shown in many investigations to be the major cause of various degenerative changes in SCAA formation (Nakatani et al., 1991; Stehbens, 1989). This hemodynamic stress might induce a complex, multifactorial remodeling through a variety of mediators and pathways. Recent studies have reported the role of nitric oxide in the development of SCAA. Inducible NO synthase (iNOS) was induced in response to hemodynamic stress and NO synthesized by iNOS serves to damage the arterial wall and lead to aneurysm formation (Fukuda et al,. 2000). Other molecular mechanisms such as active expressions of matrix metalloproteinases (Houghton et al., 2006), apoptosis of medial smooth muscle cells (Cohen et al., 1991) have been shown associated with SCAA. The role of elastase in the degradation of I.E.L in early aneurismal lesions has also been discussed. Nagata et al. (1981) reported that in experimental aneurysms many leukocytes were present adhering to the inter endothelial gaps, which may represent the participation of leukocytes in degradation of the I.E.L. Cajander and Hassler (1976) also found extracellular lysosome-like granules closely connected to the disintegrated elastic lamella in the mouths of aneurysms and hypothesized that discharged leukocyte granules containing elastase help to destroy the elastic lamella. Enhanced activity of elastase in the arterial wall may also participate in the degenerative changes of the internal elastic lamina, as in the case of hypertension (Yamada et al., 1983).
Two studies have brought significant insights into the mechanism of formation of cerebral aneurysms.
1. Futami et al. (1995) have demonstrated that fibronectin (as well as collagen IV and I) normally expressed in the subendothelium of artery, disappears in early aneurysmal lesions. The absence of fibronectin in the aneurysm wall is a critical feature in aneurysm formation considering the role of this Extra-cellular Matrix (ECM) protein in wound repair and its role in modulation of SMC phenotype (see below).
2. Jamous et al. (2007) have demonstrated the sequence of ultrastructural, morphological and pathological changes leading to the formation of saccular intracranial aneurysms in vivo. They used the current established animal model to induce cerebral aneurysm. They studied the anterior cerebral artery-olfactory artery bifurcation morphologically by using vascular corrosion casts and immunohistochemically by using specific antibodies against endothelial nitric oxide synthase (eNOS), α-smooth muscle actin (α-SMA: marker of SMCs), macrophages, and matrix metalloproteinase-9. They showed that the formation of intracranial aneurysms starts with endothelial injury at the apical intimal pad (evidenced by the loss of eNOS expression) (stage I); this leads to the formation of an inflammatory zone. This inflammatory zone shows subendothelial expression of α-SMA and a loss of eNOS. There is no protusion on the vessel wall at this early inflammatory stage (stage IIA). The progression of inflammation results in arterial wall destruction and the development of a defect presenting as a narrow slit; this is associated with protusion of the vessel wall (Stage IIB). This defect is continuous with the lumen of the parent artery, lacks eNOS expression, and contained α-SMA positive SMCs and macrophages. Expansion of this defect results in the formation of a saccular dilatation (stage III). The walls of the cavity continued to lack eNOS expression, contained a layer of α-SMA-positive SMCs and are positive for MMP-9 expression. The authors suggested that endothelial injury and exposure of the subendothelial matrix initiate platelet activation and adhesion. Activated platelets secrete growth factors that contribute to the recruitment of macrophages and promote migration of SMCs. These processes result in the formation of the inflammatory zone. The combined effects of hemodynamic changes and the destructive effects of macrophages through their release of proteolytic enzymes may lead to development of the defect.
Scanning electron microscopy studies of vascular corrosion casts of the ACA-OA bifurcation and double immunostaining of the vascular wall illustrates that normal endothelial cells are seen at the apical intimal pad, and the endothelial cell markings are elongated in the direction of the blood flow. The endothelial and the smooth-muscle layer form two continuous layers. In stage I, there are roughened apical intimal pad with irregularly shaped imprints, and loss of eNOS expression at the apical intimal pad is observed. In stage IIA, there is shallow elevation surrounded by an area of depression of the apical intimal pad. Swelling of the vessel wall at the apical intimal pad is shown, and part of this swollen area lacks eNOS expression and shows subendothelial expression of α-SMA-positive cells. In stage IIB, there is pyramid-shaped elevation of the apical intimal pad, and the surface of this elevation is covered by abnormal imprints. Thinning and degradation of the smooth-muscle layer creates a defect in the inflammatory zone (arrow) and produces vessel wall protrusion. In stage III, there is saccular aneurysm covered with abnormal imprints, expansion of the inflammatory zone defect, and destruction and protrusion of the vessel wall representing the nidus of the cerebral aneurysm.
Stage IIA has early inflammatory changes characterized by SMC migration and macrophage infiltration. A sagittal cut of the left ACA-OA bifurcation viewed at low and high magnification shows swelling of the apical intimal pad. Double immunostaining of an ACA-OA section with eNOS antibodies and α-SMA shows swelling of the vessel wall at the apical intimal pad; part of this swollen area lacks eNOS expression and shows migration of a-SMA-positive cells into the neointima. In triple immunostaining of an ACA-OA section with antibodies against eNOS, α-SMA, and macrophages, macrophage expression confirms the presence of an inflammatory zone.